Lactic acid bacteria, antibacterial substance produced by the bacteria, fermented milk starter containing the bacteria, and process for producing fermented milk by using the starter

ABSTRACT

Disclosed herein are Streptococcus salivarius subsp. thermophilus and variants thereof capable of producing an antibacterial substance which is a peptide or protein or a conjugate thereof, the antibacterial substance produced by the bacteria, and processes for producing fermented milk by using any one of the bacteria as a starter. The lactic acid bacteria produce the antibacterial substance, so that use of any one of the lactic acid bacteria as a starter for fermented milk inhibits growth of another lactic-acid-foxing lactic acid bacteria also used as a starter owing to the antibacterial substance produced during fermentation. It is therefore possible to suppress the formation of the acid during storage or transportation of fermented milk, thereby making it possible to prevent taste and flavor variations and quality deterioration of the product.

This is a division of application Ser. No. 07/658,031 filed Feb. 22,1991, abandoned.

BACKGROUND OF THE INVENTION

1) Field of the Invention

The present invention relates to novel lactic acid bacteria belonging tothe genus Streptococcus salivarius subsp. thermophilus and variantsthereof.

This invention is also concerned with a novel antibacterial substanceproduced by the above lactic acid bacteria or any one of the abovevariants.

Further, the present invention pertains to fermented milk starterscomprising the above lactic acid bacteria or any one of the abovevariants and also to processes for producing fermented milk whoseacidity increase during storage or transportation is reduced, saidprocesses making use of any one of the fermentation starters.

2) Description of the Related Art

A variety of thermophilic bacteria are found in milk and milk products.They are lactic acid bacteria useful as starters for yogurt or cheese.Among these, some thermophilic bacteria are known to produce a lowmolecular, antibacterial substance having a molecular weight not higherthan 700 [Pulusani, S.R., et al., "Journal of Food Science", 44, 575(1979)]. However, there have not been known yet any thermophilicbacteria which produces a high-molecular weight, antibacterial substancecomposed of a protein or peptide or a conjugate thereof as in thepresent invention.

The term "thermophilic bacteria" as used herein is a generic term forthe known bacteria which belong to the genus Streptococcus salivariussubsp. thermophilus. On the other hand, the term "antibacterialsubstances" as employed herein does not mean the known low-molecularweight compounds having antibacterial activities such as organicacids--for example, lactic acid--and hydrogen peroxide but is a genericterm for substances composed of a peptide, a protein or a conjugatethereof and having antibacterial activities.

In general, antibacterial substances produced by lactic acid bacteriaare used to enhance the storability of foods or to avoid deteriorationin quality of foods due to their contamination by saprophytes, astypified by nisin produced by certain Lactococcus lactis subsp. lactis(formerly, Streptococcus lactis subsp. lactis).

In the case of a fermented food containing viable cells therein, it isextremely difficult to maintain its favorable taste and flavor, whichthe fermented food has immediately after its production, continuouslyduring its storage and distribution period. For example, yogurt whichfeatures the inclusion of viable cells therein is accompanied by thetaste- and flavor-related drawback that its acidity increases during itsstorage and distribution period and its sour taste becomes stronger. Avariety of methods have heretofore been attempted with a view towardpreventing an acidity increase during storage of yogurt. These methodswere however all impractical because they were unable to bring aboutsufficient effects, rendered the production process extremely complexand cumbersome, or resulted in a very high production cost.

Fermented foods such as yogurt contain viable cells therein as describedabove. These viable cells remain active during storage so that they maydeteriorate the taste and flavor of such fermented foods during theirstorage.

To avoid deterioration in taste and flavor of fermented foods by such acause, it is most preferable to inhibit the activity of the bacteria,said activity being a cause for the deterioration in taste and flavor,without modifying their conventional production steps.

Yogurt contains Lactobacillus delbrueckii subsp. bulgaricus as principalstarter bacteria together with one or more thermophilic bacteria. Theformer bacteria are a primary cause for acidity increase at least duringthe storage and distribution period. It is hence possible to effectivelyavoid deterioration in quality due to acidity increase during thestorage and distribution period if the growth of the former bacteria canbe suppressed and the production of the acid by the bacteria strain canbe minimized directly.

SUMMARY OF THE INVENTION

With the foregoing in view, the present inventors proceeded with aninvestigation on possible interaction between bacteria, which can serveas starters for fermented foods, and the growth of thermophilicbacteria. In the course of the investigation, it was found that freshmilk contains thermophilic bacteria which inhibit the growth of suchstarters. The present inventors then succeeded in isolating andidentifying the thermophilic bacteria.

A further investigation of the thermophilic bacteria so isolated hasrevealed that these bacteria are novel and produce an antibacterialsubstance, leading to completion of the present invention.

An object of the present invention is to provide novel lactic acidbacteria which produce an antibacterial substance.

Another object of the present invention is to provide the antibacterialsubstance produced by the novel lactic acid bacteria.

A further object of the present invention is to provide a starter forfermented milk, said starter comprising the novel lactic acid bacteria.

A still further object of the present invention is to provide a processfor producing fermented milk by using such a starter, said fermentedmilk featuring reduced acidity increase during its storage ortransportation.

In one aspect of the present invention, there is thus provided a novellactic acid bacteria isolated from milk and belonging to the genusStreptococcus salivarius subsp. thermophilus.

In another aspect of the present invention, there is also provided anantibacterial substance available from a precipitate of ammonium sulfatefractionation by culturing the lactic acid bacteria in a liquid culturemedium and then subjecting the resultant culture to ammonium sulfatefractionation.

In a further aspect of the present invention, there is also provided astarter for fermented milk, said starter comprising the above lacticacid bacteria.

In a still further aspect of the present invention, there is alsoprovided a process for producing fermented milk by using the abovestarter, said fermented milk featuring reduced acidity increase duringits storage or transportation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 diagrammatically illustrates the antibacterial activities of aculture medium and the absorbance at the wavelength of 660 nm as afunction of the culture time of the lactic acid bacteria according tothe present invention, in which dots and circles indicate antibacterialactivities and absorbance, respectively; and

FIG. 2 diagrammatically shows an increase in acidity when the yogurtproduced in Example 5 was stored at 10° C.

DETAILED DESCRIPTION OF THE INVENTION

The novel lactic acid bacteria according to the present invention wasisolated from milk in the following manner.

Fresh milk was added to the concentration of 5% in a 10% (w/w)reconstituted skim milk medium which had been pasteurized at 115° C. for15 minutes, followed by incubation at 40°-45° C. until coagulation.After coagulation was repeated twice to three times in a similar manner,an inoculating loopful of the resultant culture was collected, smearedon the agar medium [1.0% (w/w) soytone, 0.5% (w/w) yeast extract, 1.0%(w/w) lactose, 1.0% (w/w) sodium succinate, 0.2% (w/w) potassiumdihydrogenphosphate, 0.2% (w/w) dipotassium hydrogenphosphate, 1.5%(w/w) agar powder; pH 6.8; hereinafter abbreviated as "SYL agar medium"]proposed by Irie et al. in Journal of Japanese Society of AgriculturalChemistry, 45, 423 (1971), and then cultured at 40° C. for 2-3 days.From numerous colonies so formed, strains were isolated. Using as testbacteria Lactobacillus helveticus subsp. jugurty which is used as astarter for fermented milk, the thus-isolated strains were tested by cupmethod in which SYL agar medium was used. The strains which exhibitedantibacterial activities were selected. With respect to each of the cellstrains so selected, the antibacterial activities were tested by a brothdilution method in which the culture medium [1.0% (w/w) soytone, 0.5%(w/w) yeast extract, 1.0% (w/w) lactose, 1.0% (w/w) sodium succinate,0.2% (w/w) potassium dihydrogenphosphate, 0.2% (w/w)dipotassiumhydrogenphosphate; pH 6.8; hereinafter abbreviated as "SYLmedium"] proposed by Irie et al. in "Journal of Japanese Society ofAgricultural Chemistry", 45, 423 (1971). One of the strains, on whichantibacterial activities were observed, was selected and stored.

With respect to the bacteria so selected, its mycologicalcharacteristics were tested. It was found to have the followingcharacteristics:

(a) Morphology

The following morphology is exhibited in SYL medium:

(1) Cell shape and size: Micrococci or diplococci. The cell size rangesfrom 0.8 μm to 1.0 μm.

(2) Gram staining: Positive.

(b) The following physiological properties are shown:

    ______________________________________                                        (1)   Coagulation of skim milk  +                                             (2)   Production of ammonia     -                                             (3)   Production of catalase    -                                             (4)   Growth range                                                                  (i)     Temperature                                                                   10° C. growth  -                                                       45° C. growth  +                                               (ii)    Salt resistance                                                               2% salt resistance    +                                                       6.5% salt resistance  -                                               (iii)   pH                                                                            pH 9.6 resistance     -                                               (iv)    0.1% methylene blue resistance                                                                      -                                         (5)   VP test                                                                 (6)   Carbohydrate fermentation ability:                                            (i)     Arabinose             -                                               (ii)    Xylose                -                                               (iii)   Glucose               +                                               (iv)    Mannose               +                                               (v)     Fructose              +                                               (vi)    Galactose             +                                               (vii)   Maltose               -                                               (viii)  Sucrose               +                                               (ix)    Lactose               +                                               (x)     Trehalose             -                                               (xi)    Sorbitol              -                                               (xii)   Mannitol              -                                               (xiii)  Glycerol              -                                               (xiv)   Starch                -                                               (xv)    Raffinose             -                                               (xvi)   Salicin               -                                               (xvii)  Cellobiose            -                                               (xviii) Dextrin               -                                               (xix)   Inulin                -                                         (7)    Production of antibacterial substance                                                                  +                                             ______________________________________                                    

Based on the above mycological characteristics, the above bacteria wereclassified in accordance with Bergey's Manual of SystematicBacteriology, Vol. 2 (compiled jointly by P.H.A. Sheath, N.S. Mair, M.E.Sharpe and J.G. Holt, pp 1069-1070, William & Wilkins, Baltimore U.S.A.,1986). As a result, the bacteria was identified as bacteria belonging tothe genus Streptococcus salivarius subsp. thermophilus and, in view ofthe production of the antibacterial substance, as a novel straindifferent from the conventional bacteria.

The present inventors therefore named this bacteria as Streptococcussalivarius subsp. thermophilus SBT 1277, and deposited Dec. 11, 1989with Fermentation Research Institute, Agency of Industrial Science andTechnology, Ministry of International Trade and Industry, Government ofJapan, 8-1, 5-chome, Inagehigashi, Chiba, Japan to which the depositnumber FERM P-11155 was allotted. The deposit of the bacteria was thentransferred to international deposit, so that FERM BP-3234 was allotted.

This bacteria can be transformed by general transformation treatment,for example, radiation such as ultraviolet rays or a chemical reagentsuch as N-methyl-N'-nitro-N-nitrosoguanine (NTG). Such variants of thesebacteria are embraced by the present invention.

This invention also relates to the antibacterial substance produced bythe above lactic acid bacteria according to the present invention or byany one of the above variants of the present invention. Theantibacterial substance according to the present invention is a peptideor protein or a conjugate thereof, which can be obtained from aprecipitate fractionated with ammonium sulfate at 30-70% saturation byculturing the lactic acid bacteria or variant in SYL medium, forexample, at 30° C. for 24 hours and subjecting the resultant culture toammonium sulfate fractionation.

The antibacterial spectrum of the antibacterial substance according tothe present invention is shown in Table 1.

                  TABLE 1                                                         ______________________________________                                                                   MIC*                                               Bacteria tested            (units/ml)                                         ______________________________________                                        Bacillus cereus SBT 3051    >32                                               Bacillus strearorthermophilus SBT 3012                                                                     1                                                Bacillus subtilis SBT 3011  32                                                Bifidobactereria longum SBT 2928                                                                           8                                                Bifidobacteria breve subsp. breve SBT 2803                                                                 4                                                Escherichia coli SBT 3057   >32                                               Lactococcus lactis subsp. lactis SBT 1219                                                                 >32                                               Lactococcus lactis subsp. diacetylactis SBT 1255B                                                         >32                                               Lactococcus lactis subsp. cremoris SBT 1323                                                               >32                                               Lactobacillus acidophilus SBT 2062                                                                        32                                                Lactobacillus delbrueckii subsp. bulgaricus MRC-32-R                                                       4                                                Lactobacillus casei SBT 2209                                                                              >32                                               Lactobacillus helveticus subsp. jugurti SBT 2161                                                           1                                                Pseudomonas aeruginosa SBT 3267                                                                           32                                                Pseudomonas fluorescens SBT 3224                                                                          16                                                Pseudomonas fragi SBT 3227  32                                                Salmonella typhimurium SBT 3075                                                                           >32                                               Sarcina lutea SBT 3013      >32                                               Serratia marcescens SBT 3155                                                                              >32                                               Staphylococcus aureus SBT 3159                                                                             4                                                Streptococcus faecalis SBT 1120                                                                           >32                                               Streptococcus faecium SBT 1155                                                                            >32                                               Streptococcus mutans SBT 1454                                                                             >32                                               Streptococcus salivarius subsp. thermophilus SBT 0137                                                      2                                                ______________________________________                                         *MIC: Minimum inhibitory concentration.                                       1 Unit: Weight of the antibacterial substance required to inhibit growth      of 10.sup.6 cells of Lactobacillus helveticus subsp. jugurty.            

As is understood from Table 1, the antibacterial substance showsantibacterial activities against gram-positive bacteria only and has anarrow antibacterial spectrum, so that the antibacterial substanceexhibits properties of typical bacteriocins.

The antibacterial spectrum of Table 1 was prepared by the followingmethod.

On a commercially-available, sterilized 96-well microplate, 200 μlportions of a liquid medium inoculated with 1% of one of the bacteria tobe tested, said medium being suited for the growth of the bacteria, wereadded to and mixed with 50 μl portions of samples of the doublingdilution system of the precipitate fractionated with ammonium sulfate at30-70% saturation, respectively. The bacteria were then cultured atpredetermined temperature suitable for the growth of the particularbacteria. The growth of the particular bacteria in each culture wasqualitatively judged. The lowest one of the concentrations of theammonium-sulfate-fractionated precipitate added to the cultures in whichthe growth was suppressed was recorded as the minium inhibitoryconcentration.

In addition, the thermal stability of the above-describedammonium-sulfate-fractionated precipitate and its digestion by trypsinand phospholipase D were measured. The measurement data were comparedwith the corresponding data of conventional bacteriocins (nisin,lactostrepcin, and diplococcin) known to be produced by streptococci.The results are summarized in Table 2.

                  TABLE 2                                                         ______________________________________                                                 Properties of antibacterial substance                                                      Phospho- Heat                                                      Trypsin    lipase D treatment                                      Antibacterial                                                                            digest-    digest-  (100° C.,                               substance  ability    ability  30 min, pH 8.0)                                ______________________________________                                        Antibacterial                                                                            S*         R**      R                                              substance of                                                                  the invention                                                                 Nisin      R          --       S                                              Lactostrepcin                                                                            S          S        --                                             Diplococcin                                                                              S          --       S                                              ______________________________________                                         *S: sensitive.                                                                **R: resistant.                                                          

As is readily envisaged from Table 2, the anti-bacterial substance ofthe present invention shows different properties in heat treatment,trypsin digestability and phospholipase D digestability compared withthe conventionally-known bacteriocins produced by streptococci.Accordingly, the antibacterial substance of the present invention can beundoubtedly judged as a novel substance.

Incidentally, the heat treatment test, the trypsin digestability testand the phospholipase D digestability test were conducted by thefollowing methods.

1) Heat resistance test

The precipitate fractionated with ammonium sulfate was dissolved in abuffer of pH 8.0 and then heated for 30 minutes in a boiling bath. Afterthe solution was cooled, the antibacterial substance was determined"resistant" or "sensitive" depending on the "presence" or "absence" ofan inhibited zone in accordance with the cup method* in whichLactobacillus helvecticus subsp. jugurty was used as an indicator strainof an antibacterial test.

2) Trypsin digestability and phospholipase D digestability

The precipitate fractionated with ammonium sulfate was dissolved in abuffer of pH 7.0. Portions of the resultant solution were added with theenzymes, trypsin and phospholipase D, respectively to give the finalconcentration of 5 mg/ml. The resulting mixtures were incubated at 25°C. for 1 hour. Using the cup method* in which Lactobacillus helveticussubsp. jugurty was used as test bacteria, the antibacterial substancewas determined "resistant" or "sensitive" depending on the "presence" or"absence" of an inhibited zone.

A cup cylinder is placed on an agar plate in which Lactobacillushelveticus subsp. jugurty has been mixed and diluted in advance(inocultun size: 1%). A sample (100 μl) is placed in the cup cylinder,followed by incubation at 37° C. for 16 hours. The diameter of aninhibited zone is then measured.

The antibacterial substance according to the present invention can beobtained by the process described above. It is a peptide or protein or aconjugate thereof, is digested by trypsin but not by phospholipase D,and exhibits stability even after heated at 100° C. and pH 8.0 for 30minutes.

It is to be noted that mixtures comprising the antibacterial substanceare also embraced by the present invention. Exemplary mixtures whichcomprise the antibacterial substance include culture media, cultures andsupernatants thereof, and the like.

The antibacterial substance of the present invention and mixturescomprising the same can be used as preservatives for beverages, foods,feed and the like, if necessary, after application of sterilizationtreatment.

This invention also relates to starters for fermented milk, saidstarters comprising the lactic acid bacteria of the present invention orany one of the variants of the present invention and also to fermentedmilk obtained using such starters.

According to the present invention, the above lactic acid bacteria isinoculated to and cultured in sterilized skim milk. The resultantculture is used as a starter for fermented milk. Combined use of thisstarter for fermented milk together with lactic acid bacteria heretoforeemployed as a starter for yogurt, for example, thermophilic bacteria,Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus helveticussubsp. jugurty or Bifidobacteria breve subsp. breve makes it possible toinhibit the growth of the bacteria by the antibacterial substanceproduced by the starter. It is therefore possible to reduce qualityreduction of a fermented food containing one or more of such strainstherein, which quality reduction is caused by excessive production ofthe acid during the storage and distribution period of the fermentedfood. Especially, among thermophilic bacteria and Lactobacillusdelbrueckii subsp. bulgaricus which are commonly employed as startersfor fermented milk, the growth of the latter bacteria as cause forquality deterioration during a storage and distribution period can besuppressed. It is therefore possible to improve the quality of fermentedmilk.

In addition, the strain of the lactic acid bacteria and its variantsaccording to the present invention can be used not only as starters foryogurt but also as fermentation regulators, for example, to improve thequality of milk products such as sour milk beverages and fermented milk.The starters according to the present invention, which are suited foruse in the production of fermented milk, can also be used as suchfermentation regulators.

Illustrative of the fermented milk which can be produced by the processof the present invention include yogurt (both hard-type and soft-type)and sour milk beverages. Upon production of fermented milk in thepresent invention, the lactic acid bacteria of the present invention andone of the above-described conventional lactic acid bacteria--which havebeen employed for the production of fermented milk--are inoculated asstarters either individually and successively or in combination to a rawmaterial for fermented milk, followed by fermentation. The raw materialfor the fermented milk, the amounts of the starters and the conditionsfor the fermentation can chosen as in the conventional processes.

The present invention will hereinafter be described more specifically bythe following examples. It should however be borne in mind that thepresent invention is not limited to or by the following examples.

EXAMPLE 1

SYL medium (50 l) was sterilized at 121° C. for 15 minutes and thencooled to 30° C. A precultured broth (500 ml) of the lactic acidbacteria according to the present invention was inoculated to themedium, followed by incubation at 30° C. for 24 hours. After theincubation, the resultant culture was cooled to 4° C. Cells werecollected at 3,000 rpm by a centrifugator, whereby the lactic acidbacteria of the present invention was obtained (yield: 1,174 g as wetcells).

EXAMPLE 2

SYL medium (16 l) was sterilized at 121° C. for 15 minutes and thencooled to 30° C. A precultured broth (160 ml) of the lactic acidbacteria according to the present invention,antibacterial-substance-producing bacteria, was inoculated to themedium, followed by incubation at 30° C. for 24 hours. Immediately afterthe incubation, the resultant culture was cooled to 4° C. Cells wereremoved at 3,000 rpm by a centrifugator, whereby a supernatantcontaining the antibacterial substance was obtained (yield: 14.9 l). Itsantibacterial activities were 8 units per ml so that the overallantibacterial activities were 119,200 units. Incidentally, 1 unit isdefined as the weight of the antibacterial substance required to inhibitgrowth of 10⁶ cells of Lactobacillus helveticus subsp. jugurty.

In the present invention, a relationship between culture time and theantibacterial activities of a culture can be expressed as shown inFIG. 1. The antibacterial activities abruptly increased after 6 hours ofincubation and reached a maximum around 8 hours of incubation. Thisbehavior was slower compared with the absorbance at the wavelength of660 nm, which indicated the amount of cells.

EXAMPLE 3

Sterilized at 121° C. for 15 minutes was Elliker agar medium [2% (w/w)tryptone, 0.5% w/w yeast extract, 0.25% (w/w) gelatin, 0.5% (w/w)dextrose, 0.5% (w/w) lactose, 0.5% (w/w) sucrose, 0.4% (w/w) sodiumchloride, 0.15% (w/w) sodium acetate, 0.05% (w/w) ascorbic acid, 1.5%(w/w) agar; pH 6.8]. While the medium was in a liquid state, thesterilized medium was poured as 20 ml portions in sterilized Petridishes. The medium was then cooled and solidified, whereby agar plateswere prepared. The lactic acid bacteria of the present invention, whichhad been cultured in SYL medium, was smeared in the form of a line by aninoculating loop on each of the agar plates, and was then cultured at30° C. for 24 hours to allow it to produce the antibacterial substance.The agar plates were exposed for 1 hour to ultraviolet rays from anultraviolet sterilization lamp, so that the bacteria thus smeared wassterilized. Mixtures of 0.1 ml of test bacteria cultured in Ellikermedium and Elliker soft agar medium [a culture medium obtained byadjusting the concentration of agar of Elliker agar medium to 0.7%(w/w)] cooled to 45° C. subsequent to its melting were placed as a layerover the agar plates, respectively. The test bacteria consisted of 45thermophilic bacteria strains used as starters for fermented milk and 27Lactobacillus delbrueckii subsp. bulgaricus strains. The thus-overlayedagar plates were incubated at 37° C. for 24 hours, followed by thedetermination of the presence or absence of an inhibited band. As aresult, such an inhibited band was observed on 43 thermophilic bacteriastrains and 7 Lactobacillus delbrueckii subsp. bulgaricus strains.

EXAMPLE 4

The lactic acid bacteria of the present invention were inoculated inpasteurized skim milk and then cultured to provide Starter A forfermented milk. A mixed starter of commercial thermophilic bacteria andLactobacillus delbrueckii subsp. bulgaricus were inoculated inpasteurized skim milk and then cultured to provide Starter B forfermented milk. Both the starters were inoculated to the concentrationof 1.5%, each, to the same pasteurized yogurt mix (pasteurized milkenriched with powdered skim milk), followed by incubation at 42° C.until the lactic acid acidity reached 0.8%. The culture was immediatelycooled, whereby the culture containing the lactic acid bacteria of thisinvention was obtained as yogurt.

The yogurt of this example was free from an acidity increase after itsproduction, thereby permitting stable storage.

EXAMPLE 5

Streptococcus salivarius subsp. thermophilus SBT 1277 according to thepresent invention and Lactobacillus delbrueckii subsp. bulgaricusSBT-2118B (MRC-32-RO) were inoculated to the concentration of 1.5%,each, to a yogurt mix which consisted of fresh milk, powdered skim milkand sugar, followed by incubation at 42° C. until the acidity reached0.75% so that yogurt was obtained. The yogurt was refrigerated at 10° C.and stored for 14 days. The acidity increase during the storage periodis plotted in FIG. 2.

As is understood from FIG. 2, substantially no acidity increase wasobserved at 10° C.

The present invention provides novel lactic acid bacteria belonging tothe genus Streptococcus salivarius subsp. thermophilus. The novel lacticacid bacteria according to the present invention produce anantibacterial substance of the bacteriocin type. This antibacterialsubstance can be used as a preservative for beverages, foods, feed andthe like.

Further, fermented milk can be produced using the lactic acid bacteriaof the present invention as a starter. Especially, its use as a starterfor yogurt can suppress production of the acid during the storage andtransportation of the resultant yogurt, thereby making it possible toprevent quality reduction of the yogurt due to excessive production ofthe acid.

What is claimed is:
 1. An isolated and purified lactic acid bacteria Streptococcus salivarius subsp. thermophilus, SBT 1277 (FERM BP-3234), or a variant thereof produced by transforming the bacteria by ultraviolet ray radiation treatment or by treatment with N-methyl-N'-nitro-N-nitrosoguanine, which isolated and purified bacteria or variant thereof, when cultured in a liquid culture medium, produces an antibacterial substance having the following properties:(a) said substance is a peptide or protein, or a conjugate thereof, (b) said substance is digested by trypsin, (c) said substance is not digested by phospholipase D, and (d) said substance exhibits thermal stability even after being heated at pH 8.0 and 100° C. for 30 minutes.
 2. A fermented milk starter comprising the lactic acid bacteria, Streptococcus salivarius subsp. thermophilus or variant thereof of claim
 1. 3. A fermented milk starter comprising the variant of the bacteria of claim
 1. 